Difference between revisions of "Part:BBa K4390105"

Latest revision as of 13:54, 12 October 2022

arsR controlled Broccoli biosensor

Improvement of part BBa_K3380600

This part is an improvement of Part:BBa_K3380600, as it uses a brigther aptamer. Broccoli binds DFHBI more tightly than iSpinach, hence producing brighter fluorescence (See Figure 1).

The Edinburgh-UHAS_Ghana team for 2022 designed a construct to detect arsenic in contaminated water. This construct is composed of a fluorescent RNA aptamer Broccoli flanked by the F30 tRNA scaffolds (BBa_K4390095) which under a strong T7 RNA promoter (BBa_I712074), downstream of the promotor there is the arsenic promotor which acts as the binding site for the arsR repressor (BBa_K346002) as to allow transcriptional repression of the T7 promotor. This construct is designed to be cell-free and only requires transcription of the RNA aptamer to produce fluorescence. It should be noted that T7 RNA polymerase, chemical energy (ATP), NTPs and DFHBI or DFHO are also required in the cell-free reaction so that fluorescence is observed.

Usage and Biology

This biosensor should be used with a cell-free lysate which contains the arsR repressor T7 RNA polymerase, chemical energy (ATP), NTPs and DFHBI or DFHO to induce fluorescence in the presence of arsenic ions. The arsR repressor will bind to the arsR binding site on the arsenic promotor when arsenic ions are not present in the reaction. This causes transcriptional repression of the RNA aptamer as the T7 polymerase is physically occluded from reading the linear construct. When arsenic ions are present in the reaction the pbrR repressor will then bind to the arsenic ions and it will allow the transcription of the RNA aptamer, this aptamer can then bind to the DFHBI or DFHO which will induce fluorescence.

Figure 1: The brightness of various aptamers in combination with various fluorophores. The column labelled "Green" represents iSpinach. Citation: Millacura FA, Li M, Valenzuela-Ortega MA, French C. TXO: Transcription-Only genetic circuits as a novel cell-free approach for Synthetic Biology. bioRxiv. 2019.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal SpeI site found at 52
12
INCOMPATIBLE WITH RFC[12]
Illegal SpeI site found at 52
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal SpeI site found at 52
25
INCOMPATIBLE WITH RFC[25]
Illegal SpeI site found at 52
1000
COMPATIBLE WITH RFC[1000]

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 ==Improvement of part BBa_K3380600==
-This part is an improvement of [[Part:BBa_K3380600]], as it uses a brigther aptamer. We designed a TXO ArsR-regulated T7 promoter biosensor expressing Broccoli instead of iSpinach ([[Part:BBa_K4390105]]). Broccoli binds DFHBI more tightly than iSpinach, hence producing brighter fluorescence (See Figure 1).
+This part is an improvement of [[Part:BBa_K3380600]], as it uses a brigther aptamer. Broccoli binds DFHBI more tightly than iSpinach, hence producing brighter fluorescence (See Figure 1).
+The Edinburgh-UHAS_Ghana team for 2022 designed a construct to detect arsenic in contaminated water. This construct is composed of a fluorescent RNA aptamer Broccoli flanked by the F30 tRNA scaffolds (BBa_K4390095) which under a strong T7 RNA promoter (BBa_I712074), downstream of the promotor there is the arsenic promotor which acts as the binding site for the arsR repressor (BBa_K346002) as to allow transcriptional repression of the T7 promotor. This construct is designed to be cell-free and only requires transcription of the RNA aptamer to produce fluorescence. It should be noted that T7 RNA polymerase, chemical energy (ATP), NTPs and DFHBI or DFHO are also required in the cell-free reaction so that fluorescence is observed.
+===Usage and Biology===
+This biosensor should be used with a cell-free lysate which contains the arsR repressor T7 RNA polymerase, chemical energy (ATP), NTPs and DFHBI or DFHO to induce fluorescence in the presence of arsenic ions. The arsR repressor will bind to the arsR binding site on the arsenic promotor when arsenic ions are not present in the reaction. This causes transcriptional repression of the RNA aptamer as the T7 polymerase is physically occluded from reading the linear construct. When arsenic ions are present in the reaction the pbrR repressor will then bind to the arsenic ions and it will allow the transcription of the RNA aptamer, this aptamer can then bind to the DFHBI or DFHO which will induce fluorescence.
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